| The gene expression levels of plant are regulated by various signal transduction mechanism. As an important component of the signal transduction of transcription factors play an important role in abiotic and biotic stresses, the C2H2-type zinc finger protein (ZFP) has been studied in recent years. Based on the high degree of adaptability of Mikania micrantha, MmZFP1was cloned from Mikania micrantha, and was characterized its function about disease and drought resistance. Mikania micrantha has a higher capacity of resistance to plant pathogen. In order to study the function of antifungal protein genes, MmTLP1and MmTLP2were cloned and characterized from Mikania micrantha. These results were as follows:1. C2H2-type ZFP is a kind of important transcription factor family, which is involved in transcription regulation, stress responses and other physiological processes. In the present study, MmZFP1, a two-fingered C2H2-type ZFP gene was cloned and characterized from Mikania micrantha. The results showed that MmZFP1located in nucleus and had the typical protein structure characteristics of C2H2-type ZFP. MmZFP1was expressed in roots, stems and leaves. It was strongly expressed in roots, but weakly in stems and leaves. Expression of MmZFP1could be up-regulated by Verticillium dahliae (Vd991) infection, SA, ethylene, ABA, dehydration and high salinity. The observations of phenotypes indicated that constitutive expression of MmZFPl in transgenic plants resulted in growth and development retardation that was correlated with the expression level of MmZFP1. MmZFP1transgenic Arabidopsis thaliana significantly increase the resistance to Pst.DC3000and Vd991. Transgenic Arabidopsis thaliana plants could up-regulate the expression levels of defense genes including PR1, PR2under normal growing conditions or Vd991infection. Overexpression of MmZFP1in Arabidopsis thaliana could up-regulate the expression levels of the drought stress tolerance genes including ABF3, ABF4under normal growing conditions, which resulted in improved tolerance to drought. Thus, our results suggest that MmZFP1plays a key role in disease and drought stress resistance.2. In the present study, protein components against Valsa mali were isolated from seeds of Mikania micrantha, and using proteomics approach to analyze the components and obtaining the peptide sequence of MmTLP1. Subsequently, MmTLP1and MmTLP2, two thaumatin-like protein (TLP) genes were cloned and characterized from Mikania micrantha.The MmTLP1and MmTLP2expression patterns of Mikania micrantha displayed the expression levels of MmTLP1are similar in stem, root and leaf, while more abundant mRNA of MmTLP2in leaf than root and stem. Expression of MmTLP1could be significantly induced by Vd991infection, SA, MeJA, ethylene, ABA, dehydration and high salinity in Mikania micrantha, and expression of MmTLP2could be induced by Vd991infection, SA, MeJA, ABA, dehydration and high salinity, which indicates that MmTLP1and MmTLP2may play an important role in response to biotic and abiotic stresses. The MmTLP1and MmTLP2protein were located in the extracellular space/cell wall by MmTLP1::GFP and MmTLP2:;GFP fusion protein in transgenic Arabidopsis thaliana. MmTLP1and MmTLP2recombinant proteins could inhibit the hypha growth of Valsa mali, Botrytis cinerea and Fumrium oxysporum, which showed certain antifugal activity. Overexpression of MmTLP1enhanced the resistance of Arabidopsis thaliana against Vd991. The transgenic MmTLP2Arabidopsis thaliana had enhanced resistance against Vd991at early post-inoculation stage, but of which was not different with wild type during later infection stages. These results implicate the MmTLPl protein may be capable of protecting plants against fungal diseases, and play key role in plant defense response against pathogens. Overexpression of MmTLP1and MmTLP2in transgenic Arabidopsis thaliana significantly enhanced the tolerance to drought and high salinity stress. These results implicate the MmTLP1and MmTLP2could play key roles in abiotic stress resistance. |
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